Chapter 9 Regulation of Human Dihydrofolate Reductase Activity and Expression
Introduction
The enzyme dihydrofolate reductase (DHFR, 5,6,7,8‐tetrahydrofolate:NADP+ oxidoreductase, EC 1.51.3) catalyzes the reduction of dihydrofolate (H2F) to tetrahydrofolate (H4F) utilizing NADPH as a cofactor. H4F and its derivatives are essential cofactors in the synthesis of thymidylate, purines, and some amino acids (Figure 9.1, Figure 9.2) (Blakley and Cocco, 1984, Futterman, 1957, Osborn and Huennekens, 1958). Inhibition of DHFR results in a depletion of the reduced folate pools, inhibition of DNA synthesis, and cell death. Due to its biological significance, DHFR has proven to be an important target of antineoplastic, antiprotozoal, antifungal, and antimicrobial drugs in addition to its use for the treatment of other nonmalignant diseases, such as arthritis.
Antifolates are the oldest of the antimetabolite class of anticancer drugs and have been used in the clinic for more than four decades. The first clinically useful antifolate was aminopterin, a tight binding inhibitor of DHFR. Treatment with aminopterin led to the first‐ever remissions in childhood leukemia. Soon after, methotrexate (MTX) replaced aminopterin based on animal studies showing that MTX had a better therapeutic index.
Over the following years, in order to develop better antifolates, a detailed understanding of DHFR at every level has been undertaken such as structure–functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR using a wide range of technologies. Because of this wealth of information created, DHFR has been used extensively as a model system for enzyme catalysis, investigating the relations between structure in silico structure‐based drug design, transcription from TATA‐less promoters, regulation of transcription through the cell cycle, and translational autoregulation.
In this review, the current understanding of human DHFR is summarized. We begin with the structure and kinetic mechanism of enzyme of DHFR. The review then concentrates on the genomic organization, polymorphisms, transcriptional, and translational regulation of DHFR. We refer readers to earlier reviews that discuss many aspects of DHFR regulation for additional in‐depth analysis (Azizkhan et al., 1993, Banerjee et al., 2002, Blakley, 1995, Cody et al., 2006, Hammes‐Schiffer and Benkovic, 2006, Schnell et al., 2004, Slansky and Farnham, 1996).
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Structure and Binding of Dihydrofolate, MTX, and NADPH
As a result of its importance, a detailed picture how DHFR works emerged by studying its kinetics and structure; however, the most extensive structural characterization has been done for Escherichia coli. Fortunately, the amino acids required for catalysis and the general features of the secondary structures have been conserved throughout the evolution expect in protozoa and plants where DHFR is fused to thymidylate synthase as a bifunctional enzyme.
DHFR shares a modified version of a common
Mechanism of DHFR Catalysis
DHFR catalyzes the reduction of 7,8‐dihydrofolate to 5,6,7,8‐tetrahydrofolate using NADPH as the hydride donor. Specifically, the pro‐R hydrogen of NADPH is transferred to C6 of the pteridine ring with concomitant protonation at the N5 position (Fig. 9.2A) (Benkovic and Hammes‐Schiffer, 2003). The kinetic mechanism of E. coli and human DHFR catalysis are studied extensively by several groups (Blakley, 1995, Hammes‐Schiffer and Benkovic, 2006, Sawaya and Kraut, 1997, Schnell et al., 2004).
Alternative Substrates: Folic Acid and Dihydrobiopterin
H2F and its polyglutamylate forms are the major substrate of DHFR, although the fully oxidized folate and biopterin are poor substrates of DHFR. In January 1998, Food and Drug Administration initiated the folic acid fortification program, which requires addition of 0.43–1.4 mg folic acid per pound to enriched flour. For folic acid to be converted to the physiologically useful form found in the blood stream, 5‐methyltetrahydrofolate, folic acid is reduced to H4F in the upper small intestine or
Genomic Organization of DHFR
The functional DHFR gene (GeneID: 1719) is located at chromosome 5q11.2‐q13.3. It was cloned, mapped, and sequenced by the Nienhuis and Attardi laboratories (Chen et al., 1982, Chen et al., 1984, Masters and Attardi, 1983, Maurer et al., 1984, Maurer et al., 1985). The DHFR gene is ∼30 kb long and contains six exons and five introns with strictly conserved intron/exon boundaries. The approximate length of the introns varies between 0.35 kb (intron 1) and 11.4 kb (intron 3). After pre‐mRNA
Human Dihydrofolate Reductase Pseudogenes
There is one functional DHFR gene located in the region of q11.1‐q13.3 region of chromosome 5 and at least four pseudogenes interspersed on several chromosomes. Three of the DHFR pseudogenes are located on chromosomes 3, 6, and 18 (Anagnou et al., 1984, Blakley and Sorrentino, 1998, Maurer et al., 1984, Maurer et al., 1985, Polymeropoulos et al., 1991). These pseudogenes presumably have no activity, since unlike hDHFR they were not amplified in MTX‐resistant cell lines (Anagnou et al., 1984,
Transcriptional Regulation
DHFR expression is regulated at many levels. Cellular transcription is regulated not only by the transcription factors, but also through chromatin remodeling involving acetylation and methylation of histones. Histone acetyl transferase (HAT) may be recruited to the DNA after transcriptional factor binding, thereby enhancing nucleosomal relaxation followed by increased transcription. Furthermore, transcription factors can bind directly to histone deacetylases (HDAC) that remove acetyl groups
Polymorphisms of DHFR
Single‐nucleotide polymorphisms (SNPs) are present in 1% of the human genome. SNPs might occur in coding region or noncoding region; the former may lead to defective protein due to amino acid changes, the latter may affect the gene transcription, RNA splicing, or RNA stability.
To date, no polymorphism within the coding region of DHFR have been found possibly due to the critical role of enzyme (Banerjee et al., 2002, Blakley and Sorrentino, 1998, Gellekink et al., 2007, Parle‐McDermott et al.,
Posttranscriptional Regulation of DHFR
Recently, a new posttranscriptional mechanism of gene regulation has been found in mammalian cells and plants. microRNAs (miRNA) are 22 nucleotide noncoding RNAs and act by translational repression, mRNA cleavage, mRNA deadenylation, or transcriptional silencing. miRNAs share the RNAi machinery to form hybrids with target mRNA by anchoring the 3′ end of RNA (Murchison and Hannon, 2004). The 3′UTR of DHFR harbors a mir‐24 microRNA‐binding site which is next to the SNP 829C > T (vide supra). The
Translational Regulation of DHFR
It has been over four decades since initial reports described a rapid increase in DHFR levels in response to the antifolate, MTX (Bertino et al., 1962). As increased intracellular levels of DHFR may hinder clinical success, considerable efforts and attention have been directed toward determining the exact molecular mechanism of this induction. As a result of these efforts, we and others have determined that a translational mechanism underlies this rapid increase. Recently, it has been shown
Acknowledgments
We regret that many important references could not be cited, or were cited indirectly by citing review particles due to space limitations. This work was supported by Grant CA 08010 from the United States Public Health Service (to J. R. B.) and Department of Medicine Grant from UMDNJ (to E. A. E.). We gratefully acknowledge helpful discussions with Dr. Joseph Bertino, Dr. Debabrata Banerjee, and Dr. Vivian Cody for providing the ribbon diagram of human dihydrofolate reductase.
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